Control of betaa-cell growth and survival has implications for both type-1 and -2 diabetes, but the molecular mechanisms behind the maintenance of an optimal beta-cell mass are complex and not well defined. Adult a-cell growth is further complicated in that it can be differentially contributed to by mitogenesis, survival, size, and/or neogenesis. Recently, it has become evident that IRS-2 signal transduction is key for regulation of beta-cell mass. This proposed research intends to better link IRS-2 signaling mechanisms to control of a-cell mitogenesis, survival and neogenesis. Aim-1 will develop a mechanistic connection from glucose/IGF-1 induced IRS-2 signaling to components of cell cycle control in islet beta-cells, especially in regard to a specific increase in cyclin-D2 and activation of cyclin-D dependent kinase-4 (Cdk-4) via formation of a cyclin-D/Cdk-4/p27KIP/p21CIP complex which is key to inducing a-cell mitogenesis. Aim-2 will investigate survival mechanisms in primary islet beta-cells, especially downstream of IRS-2/PKB. Activation of PKB in a-cells is protective against FFA-induced apoptosis, implicating PKB as key to beta-cell survival. However, PKB has a plethora of substrates, and not all of which are anti-apoptotic. Thus, to gain specificity of PKB-mediated prevention of a-cell apoptosis, certain PKB substrates (e.g. GSK3, Foxo1, Mdm2 and BAD) will be investigated, via adenoviral-mediated expression, for protection of beta-cells from FFA-induced apoptosis. Aim-3 will examine beta -cell neogenesis in a resurrected model, the transplantable insulinoma in the NEDH-rat. As the insulinoma grows subcutaneously, the endogenous pancreatic beta-cells atrophy but on surgical removal of the insulinoma the endogenous beta-cell population recovers, mostly by neogenesis. Intriguingly, there is a parallel emergence of IRS-2+ and insulin+ cells from proliferating ductal tissue during this beta-cell neogenesis. This model will be used to characterize a correlation between IRS-2 signaling, local growth factors and the early appearance of putative beta-cell progenitor markers (e.g. Ngn3, Pdx-1 & nestin) with insulin+ cells. Key candidate IRS-2 signaling elements and factors will emerge from this model, and these will then be examined, via adenoviral expression in pancreatic ductal epithelial cell lines, to see if insulin+ cells can be generated in vitro. From the lessons learned in aims 1-3, these will be integrated into aim-4 that examines the mechanisms for controlling a-cell mitogenesis, size, survival, and neogenesis in in vivo models of non-diabetic obesity and obesity-linked diabetes. These studies will ascertain what contribution beta-cell mitogenesis, size, survival, and neogenesis makes to a-cell growth/survival relative to control by IRS-2 signal transduction, as well as age (neonate to adult) and metabolic homeostasis of the animal. Particular attention will be paid to active IRS-2 signaling complementary to increasing beta-cell growth for adaptation to obesity-associated insulin resistance, as well as impairment of IRS-2 signaling that could contribute to a decrease in beta-cell mass that marks the onset of obesity-linked type-2 diabetes. All in all, the proposed research will give a more mechanistic and comprehensive understanding of the control of beta-cell growth that may prove insightful for generating novel therapies to treat diabetes.